| Budget Amount *help |
¥3,410,000 (Direct Cost: ¥3,200,000、Indirect Cost: ¥210,000)
Fiscal Year 2007: ¥910,000 (Direct Cost: ¥700,000、Indirect Cost: ¥210,000)
Fiscal Year 2006: ¥1,000,000 (Direct Cost: ¥1,000,000)
Fiscal Year 2005: ¥1,500,000 (Direct Cost: ¥1,500,000)
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| Research Abstract |
The endothelin family of peptides consists of four isoforms : endothelin-1 (ET-1), vasoactive intestinal contractor (VIC), endothelin-2 (ET-2) and endothelin-3 (ET-3). These endothelins (ETs) are all composed of 21-amino acid residues and, have a variety of biological roles including involvement in cell-growth activity, hormone regulation and diuresis. Recent clinical studies in humans provide convincing evidence demonstrating that ETs play pathophysiological roles in a various clinical disorders, particularly in cardiovascular, respiratory and renal disease. In spite of the accumulating information in basic or clinical medical fields, little is known about ETs in veterinary medicine. In this study, we focus on the dog, an animal in which cardiovascular disorders occur commonly with advanced age and for which cardiovascular disease is followed with much interest. Through molecular cloning and subclinical studies. on dog ETs, we show pathophysiological involvement of ETs in dog cardiova
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scular disorders.
1) cDNA cloning Molecular cloning showed that dog PPET-1 is a polypeptide of 202 amino residues, which is similar in length to human PPET 1 and pig PPET-1. Fundamental structural characteristics of PPET-1 are well conserved among mammals. PPET-2 nucleotide and deduced amino acid sequences were compared to a previously reported partial dog PPET-2 cDNA (Itoh, et al.). The following discrepancies were found between the two sequences: (1) While the cDNA we present encodes a 178-amino acid peptide showing the same structural characteristics as observed in human, mouse and rat, the cDNA by Itoh, et al. encodes a smaller PPET 2 which lacks the c-terminal portion; (2) the amino acid region corresponding to exon 1 shows little homology between our dog sequence and the sequence reported by Itoh, et al.; and (3) the nucleotide sequences corresponding to exon 1 and exon 3 reported by Itoh, et al. are more homologous to mouse intron 1 and intron 2 than to our dog exon I and exon 3 sequences. These findings suggest that the dog sequence reported by Itoh, et al. is derived from genomic DNA. The regions corresponding to big ET-2 and ET-2 like peptide as well as the target site for endothelin converting enzyme within the preproprotein, which are well conserved among human and murine animals, were found also in our dog PPET-2. Cloning of dog PPET-3 cDNA showed that dog PPET 3 is encoded by an open reading frame of 594 bp. The deduced amino acid sequence of dog PPET-3 appears to be highly conserved among mammals.
2) Pathophysiological role of ET-I in dog cardiopulmonary diseases For determining the PPET-I, PPET-2 and PPET-3 mRNA levels, a quantitative method by real-time PCR was established. Using this technique, we explored the possible involvement of ETs in cardiovascular disease from a standpoint of molecular biology. In dog affected with filariasis (heartworm), PPET-1 mRNA expression in the heart and lung are markedly increased compared with healthy dog. No significant increase in mRNA expression of PPET-2 or PPET-3 was noticed in any organ of dogs with filariasis. Moreover, plasma ET-1 concentration is significantly increased in filariasis compared with healthy controls. In addition, we investigated plasma ET-1 levels in the canine induced mitral regurgitation (MR) model as well as in dogs diagnosed with MR, ventricular septal defect (VSD), parent ductus arteriosus (PDA) and tricuspid regurgitation (TR). Elevated plasma ET-1 levels were observed in all of these cases.
In conclusion, these findings suggest that ET-1 is involved in the pathogenesis of cardiopulmonary disorders in dogs as well as in human. Furthermore, because it can be detected in the early stages of a disease, an increased plasma ET-1 level may be a useful indicator of the prognosis and progression of cardiopulmonary conditions in dog. Less
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